MOLECULAR REPRODUCTION AND DEVELOPMENT 25164-171 (1990)
Culture Media for Mouse Oocyte Maturation Affect Subsequent Embryonic Development J.J.M. VAN DE SANDT, A.C. SCHROEDER, AND J.J. EPPIG The Jackson Laboratory, Bar Harbor, Maine
ABSTRACT These experiments were done to determine whether the culture medium used for the spontaneous maturation of mouse oocytes can affect the subsequent capacity of the ova to become fertilized and complete preimplantation development in vitro and development to live young. Oocytes obtained from antral follicles of gonadotropin-primed immature mice underwent spontaneous maturation in control medium, i.e. Eagle’s Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum, or in one of eight different media which were also supplemented with serum. All of the ova were fertilized in Whitten’s medium and were assessed for cleavage to the 2-cell stage and for further preimplantation development to blastocysts during culture in Whitten’s medium. Three of the eight media used for oocyte maturation improved the capacity of the ova to develop to the blastocyst stage when compared with the control: Waymouth MB 752/1, MEM with non-essential amino acids, and MEM Alpha; Waymouth medium promoted the highest frequency of development of ova to the blastocyst stage. Moreover, the blastocysts derived from oocytes that matured in Waymouth medium contained more cells than blastocysts derived from oocytes that matured in control medium. Although BGJb medium promoted the cleavage of eggs to the 2-cell stage when present during oocyte maturation, it had a detrimental effect on their subsequent preimplantation developmental capacity. Following transfer to foster mothers, more 2-cell stage embryos developed to live young after oocyte maturation in Waymouth medium (21%) than in control medium (13%). It is concluded that the medium used for oocyte maturation in vitro can affect processes involved in the subsequent development of the eggs and that, of the media tested, Waymouth MB 752/ 1 promoted the highest capacity for embryo development of maturing mouse oocytes. Key Words: Oocyte maturation, Developmental capacity, Culture medium, Fertilization, Mouse
INTRODUCTION Oocytes isolated from mammalian antral follicles undergo gonadotropin-independent maturation in
0 1990 WILEY-LISS, INC.
vitro (Pincus and Enzmann, 1935). During this spontaneous maturation, the nuclear events, germinal vesicle breakdown (GVB) and polar body formation, appear to occur normally, but fertilization and preimplantation development after spontaneous maturation were frequently not successful (Fleming et al., 1985; Liebfried and Bavister, 1983; Thibault, 1977). It was suggested that this was caused by a n incomplete cytoplasmic maturation of the oocyte. Since fertilization and subsequent development of oocytes that matured in vivo was much more successful, it was concluded that hormonal and/or follicular factors were needed to promote cytoplasmic maturation. Nevertheless, some investigators achieved much more promising results (Cross and Brinster, 1970; Van Blerkom and McGaughey, 1978), and i t was later shown that oocytes from mice can undergo normal maturation in vitro; there were similar frequencies of fertilization, preimplantation development and production of live offspring from oocytes that matured in vivo and in vitro (Schroeder and Eppig, 1984). These results suggest that failure in some previous attempts of fertilization and further development of oocytes that underwent maturation in vitro could have been due to inadequate oocyte culture systems. Several attempts have been made to achieve a higher frequency of development after spontaneous maturation of oocytes by altering the conditions during maturation. Particular attention has been devoted to coculturing the maturing oocytes with granulosa or cumulus cells or adding hormones to the medium used for oocyte maturation (Ball et al., 1983; Schroeder et
Received May 2, 1989; accepted August 7, 1989 Address reprint requests and correspondence to John Eppig, The Jackson Laboratory, Bar Harbor, ME 04609, USA. J.J.M. van de Sandt is a Graduate Student from the Department of Animal Physiology, Agricultural University, Wageningen, The Netherlands. A.C. Schroeder is a Visiting Investigator at the Jackson Laboratory. Permanent address: Biology Department, Gettysburg College, Gettysburg, PA 17325.
CULTURE MEDIA FOR MOUSE OOCYTES al., 1988; Shalgi et al., 1979; Sirard et al., 1988; Staigmiller and Moor, 1984). I t is shown here that the nonhormonal environment in which the oocyte undergoes spontaneous maturation also plays a crucial role in the development of its capacity to undergo embryo development. Groups of cumulus cell-enclosed mouse oocytes were cultured in nine different commercially available culture media for the period of oocyte maturation and then each group was fertilized under identical conditions and allowed to undergo preimplantation development. The results show that oocytes that undergo spontaneous maturation in Waymouth MB752/1 medium develop to the blastocyst stage at a higher frequency than embryos derived from ova that matured in the other media tested. Moreover, the blastocysts derived from the ova that matured in the Waymouth medium contained more cells than the blastocysts from the other groups. Furthermore, when compared to control medium, maturation in Waymouth medium promoted the development to live young of two-cell embryos transferred to pseudopregnant foster mothers.
MATERIALS AND METHODS Animals The animals used for the experiments were (C57BLi 6J x SJL/J)F, mice. After weaning at 20 days of age, the males were caged individually while females were housed in groups of six. The age a t which the animals were used in the experiments were 22-24 days (females), and 4-6 months (males). Media The following media, purchased in powdered form from Sigma Chemical Co., were used for the maturation of oocytes: Eagle’s Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), MEM with non-essential amino acids (MEM + NEAA), Waymouth MB 752/1, BGJb medium Fitton-Jackson modification (BGJb), Nutrient Mixture F-12 (Ham), MEM Alpha, and Medium 199. As a control medium, we used the medium which we have used routinely for oocyte maturation (Schroeder and Eppig, 1984): MEM with Earle’s salts (prepared from components purchased from Sigma) and vitamins and amino acids (purchased as concentrates from M.A. BioProducts). All of the media were supplemented with penicillin G potassium salt (75 mg/liter) and streptomycin sulfate (50 mgiliter); pyruvate (0.23 mM) was added to those media that did not already contain it. All media used for maturation were also supplemented with 5% fetal bovine serum (FBS, HyClone), to prevent hardening of the zona pellucida which inhibits fertilization (Downs et al., 1986). Regardless of the medium used for oocyte maturation, ova were inseminated and subsequently cultured in the same medium, i.e. Whitten’s medium (Whitten, 19711, as described previously (Schroeder and Eppig, 1984). The water used for making the media was collected
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from a spring in Franklin (Maine) and was glass-distilled once; all media for each experiment were made from the same batch of water. Media used for oocyte maturation and Whitten’s Medium were gassed vigorously (5% COz, 5% 0, and 90% N,, Airco Medical Gasses) for 10 minutes before sterilization (0.45 pm pore Corning filter) and storage a t 4”C, and were used within two weeks of preparation.
Maturation and Fertilization of the Oocytes; Culture of the Embryos Sixteen-24 mice (20-22 days old) were injected with 5 IU pregnant mares’ serum gonadotropin (PMSG; Organon, Inc.) in phosphate buffered saline (PBS) and they were sacrificed in groups of four by cervical dislocation 44-48 h after injection of PMSG. The oocytecumulus complexes of all mice were isolated and collected in control medium within 40 min. The complexes were then washed and randomly distributed to the different media in Corning tissue culture dishes, then allowed to mature for 14-15 h. After maturation, the complexes were washed three times in Whitten’s medium, and fertilized according to the method described previously (Schroeder and Eppig, 1984). The oocytes were washed free of sperm 6-8 h after insemination and cultured in gassed borosilicate glass culture tubes containing 1 ml of Whitten’s medium. The percentage of the eggs that cleaved into two equal blastomeres was determined 24 h after insemination. The 2-cell stage embryos were cultured further in culture tubes containing fresh medium to ascertain their capacity to reach the blastocyst stage, a s described previously (Schroeder and Eppig, 1984). The percentages of the embryos that reached the 4-cell stage, morula stage and blastocyst stage were assessed a t 56,80, and 120 h after insemination respectively. The blastocysts were fixed in 4% formaldehyde in PBS, and stained with Hoechst-stain to determine the number of cells per blastocyst as described by Ebert et al. (1985). The experiment was conducted 5 times and the data were pooled for statistical evaluation using X2 analysis of the frequency a t which the ova in the various groups achieved the various stages of preimplantation development compared with the embryos derived from oocytes that matured in the control medium. To illustrate the variation between experiments, the data from key groups are presented for all 5 experiments. The number of cells per blastocyst in the various groups is presented in notched box-and-whisker plots (Kafadar, 1985) using the Stat View 512 + program from Abacus Concepts using a Macintosh I1 microcomputer. Nonoverlapping notches between a pair of samples indicates a significant difference (P
Culture Media for Mouse Oocyte Maturation Affect Subsequent Embryonic Development J.J.M. VAN DE SANDT, A.C. SCHROEDER, AND J.J. EPPIG The Jackson Laboratory, Bar Harbor, Maine
ABSTRACT These experiments were done to determine whether the culture medium used for the spontaneous maturation of mouse oocytes can affect the subsequent capacity of the ova to become fertilized and complete preimplantation development in vitro and development to live young. Oocytes obtained from antral follicles of gonadotropin-primed immature mice underwent spontaneous maturation in control medium, i.e. Eagle’s Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum, or in one of eight different media which were also supplemented with serum. All of the ova were fertilized in Whitten’s medium and were assessed for cleavage to the 2-cell stage and for further preimplantation development to blastocysts during culture in Whitten’s medium. Three of the eight media used for oocyte maturation improved the capacity of the ova to develop to the blastocyst stage when compared with the control: Waymouth MB 752/1, MEM with non-essential amino acids, and MEM Alpha; Waymouth medium promoted the highest frequency of development of ova to the blastocyst stage. Moreover, the blastocysts derived from oocytes that matured in Waymouth medium contained more cells than blastocysts derived from oocytes that matured in control medium. Although BGJb medium promoted the cleavage of eggs to the 2-cell stage when present during oocyte maturation, it had a detrimental effect on their subsequent preimplantation developmental capacity. Following transfer to foster mothers, more 2-cell stage embryos developed to live young after oocyte maturation in Waymouth medium (21%) than in control medium (13%). It is concluded that the medium used for oocyte maturation in vitro can affect processes involved in the subsequent development of the eggs and that, of the media tested, Waymouth MB 752/ 1 promoted the highest capacity for embryo development of maturing mouse oocytes. Key Words: Oocyte maturation, Developmental capacity, Culture medium, Fertilization, Mouse
INTRODUCTION Oocytes isolated from mammalian antral follicles undergo gonadotropin-independent maturation in
0 1990 WILEY-LISS, INC.
vitro (Pincus and Enzmann, 1935). During this spontaneous maturation, the nuclear events, germinal vesicle breakdown (GVB) and polar body formation, appear to occur normally, but fertilization and preimplantation development after spontaneous maturation were frequently not successful (Fleming et al., 1985; Liebfried and Bavister, 1983; Thibault, 1977). It was suggested that this was caused by a n incomplete cytoplasmic maturation of the oocyte. Since fertilization and subsequent development of oocytes that matured in vivo was much more successful, it was concluded that hormonal and/or follicular factors were needed to promote cytoplasmic maturation. Nevertheless, some investigators achieved much more promising results (Cross and Brinster, 1970; Van Blerkom and McGaughey, 1978), and i t was later shown that oocytes from mice can undergo normal maturation in vitro; there were similar frequencies of fertilization, preimplantation development and production of live offspring from oocytes that matured in vivo and in vitro (Schroeder and Eppig, 1984). These results suggest that failure in some previous attempts of fertilization and further development of oocytes that underwent maturation in vitro could have been due to inadequate oocyte culture systems. Several attempts have been made to achieve a higher frequency of development after spontaneous maturation of oocytes by altering the conditions during maturation. Particular attention has been devoted to coculturing the maturing oocytes with granulosa or cumulus cells or adding hormones to the medium used for oocyte maturation (Ball et al., 1983; Schroeder et
Received May 2, 1989; accepted August 7, 1989 Address reprint requests and correspondence to John Eppig, The Jackson Laboratory, Bar Harbor, ME 04609, USA. J.J.M. van de Sandt is a Graduate Student from the Department of Animal Physiology, Agricultural University, Wageningen, The Netherlands. A.C. Schroeder is a Visiting Investigator at the Jackson Laboratory. Permanent address: Biology Department, Gettysburg College, Gettysburg, PA 17325.
CULTURE MEDIA FOR MOUSE OOCYTES al., 1988; Shalgi et al., 1979; Sirard et al., 1988; Staigmiller and Moor, 1984). I t is shown here that the nonhormonal environment in which the oocyte undergoes spontaneous maturation also plays a crucial role in the development of its capacity to undergo embryo development. Groups of cumulus cell-enclosed mouse oocytes were cultured in nine different commercially available culture media for the period of oocyte maturation and then each group was fertilized under identical conditions and allowed to undergo preimplantation development. The results show that oocytes that undergo spontaneous maturation in Waymouth MB752/1 medium develop to the blastocyst stage at a higher frequency than embryos derived from ova that matured in the other media tested. Moreover, the blastocysts derived from the ova that matured in the Waymouth medium contained more cells than the blastocysts from the other groups. Furthermore, when compared to control medium, maturation in Waymouth medium promoted the development to live young of two-cell embryos transferred to pseudopregnant foster mothers.
MATERIALS AND METHODS Animals The animals used for the experiments were (C57BLi 6J x SJL/J)F, mice. After weaning at 20 days of age, the males were caged individually while females were housed in groups of six. The age a t which the animals were used in the experiments were 22-24 days (females), and 4-6 months (males). Media The following media, purchased in powdered form from Sigma Chemical Co., were used for the maturation of oocytes: Eagle’s Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), MEM with non-essential amino acids (MEM + NEAA), Waymouth MB 752/1, BGJb medium Fitton-Jackson modification (BGJb), Nutrient Mixture F-12 (Ham), MEM Alpha, and Medium 199. As a control medium, we used the medium which we have used routinely for oocyte maturation (Schroeder and Eppig, 1984): MEM with Earle’s salts (prepared from components purchased from Sigma) and vitamins and amino acids (purchased as concentrates from M.A. BioProducts). All of the media were supplemented with penicillin G potassium salt (75 mg/liter) and streptomycin sulfate (50 mgiliter); pyruvate (0.23 mM) was added to those media that did not already contain it. All media used for maturation were also supplemented with 5% fetal bovine serum (FBS, HyClone), to prevent hardening of the zona pellucida which inhibits fertilization (Downs et al., 1986). Regardless of the medium used for oocyte maturation, ova were inseminated and subsequently cultured in the same medium, i.e. Whitten’s medium (Whitten, 19711, as described previously (Schroeder and Eppig, 1984). The water used for making the media was collected
165
from a spring in Franklin (Maine) and was glass-distilled once; all media for each experiment were made from the same batch of water. Media used for oocyte maturation and Whitten’s Medium were gassed vigorously (5% COz, 5% 0, and 90% N,, Airco Medical Gasses) for 10 minutes before sterilization (0.45 pm pore Corning filter) and storage a t 4”C, and were used within two weeks of preparation.
Maturation and Fertilization of the Oocytes; Culture of the Embryos Sixteen-24 mice (20-22 days old) were injected with 5 IU pregnant mares’ serum gonadotropin (PMSG; Organon, Inc.) in phosphate buffered saline (PBS) and they were sacrificed in groups of four by cervical dislocation 44-48 h after injection of PMSG. The oocytecumulus complexes of all mice were isolated and collected in control medium within 40 min. The complexes were then washed and randomly distributed to the different media in Corning tissue culture dishes, then allowed to mature for 14-15 h. After maturation, the complexes were washed three times in Whitten’s medium, and fertilized according to the method described previously (Schroeder and Eppig, 1984). The oocytes were washed free of sperm 6-8 h after insemination and cultured in gassed borosilicate glass culture tubes containing 1 ml of Whitten’s medium. The percentage of the eggs that cleaved into two equal blastomeres was determined 24 h after insemination. The 2-cell stage embryos were cultured further in culture tubes containing fresh medium to ascertain their capacity to reach the blastocyst stage, a s described previously (Schroeder and Eppig, 1984). The percentages of the embryos that reached the 4-cell stage, morula stage and blastocyst stage were assessed a t 56,80, and 120 h after insemination respectively. The blastocysts were fixed in 4% formaldehyde in PBS, and stained with Hoechst-stain to determine the number of cells per blastocyst as described by Ebert et al. (1985). The experiment was conducted 5 times and the data were pooled for statistical evaluation using X2 analysis of the frequency a t which the ova in the various groups achieved the various stages of preimplantation development compared with the embryos derived from oocytes that matured in the control medium. To illustrate the variation between experiments, the data from key groups are presented for all 5 experiments. The number of cells per blastocyst in the various groups is presented in notched box-and-whisker plots (Kafadar, 1985) using the Stat View 512 + program from Abacus Concepts using a Macintosh I1 microcomputer. Nonoverlapping notches between a pair of samples indicates a significant difference (P
